JPH10505897A - Method for measuring free apoprotein (a) - Google Patents

Abstract

  (57) [Summary] It describes a method for detecting free Apo (a) in human body fluids containing free Apo (a) and Apo (a) bound to lipids, wherein the method comprises the steps of: a) is isolated and then free Apo (a) is measured in a known manner, or b) free Apo (a) is to free Apo (a) but to bound Apo (a) It is determined by an immunological reaction using a non-antibody. According to the method of the present invention, a valuable assessment of the risk of lipoprotein metabolic disease and / or atherogenesis present in a patient can be made.

Description

The present invention relates to a method for measuring free apoprotein (a) (Apo (a)). Apoprotein (a) plays an important role in the inhibition of plasminogen and other fibrinolytic proteases and in the uptake of cholesterol esters and other lipids in the arterial lining during atherogenesis. Apoprotein (a) (Apo (a)) forms lipoprotein (a) (Lp (a)) with apoprotein B 100 (Apo B-100) and lipid components. This lipid component consists of a polar lipid-encapsulated surface thin film filled with cholesterol. The lipid component with Apo B-100 attached corresponds to low density lipoprotein (LDL) (Berg et al., Acta Path. 59 (3) (1963) 369). Lp (a) is thought to be an independent risk factor in the development of atherosclerosis (Kostner et al., Atherosclerosis 38 (1981) 51), but the effects of individual apoproteins have not been elucidated. Apo (a) is a highly glycosylated protein that is thought to associate with Apo B-100 at one or more disulfide bridges (Utermann et al., J. Clin. Invest. 80 (1987) 458; Gaubatz et al., J. Biol.Chem.258 (7) (1983) 4582; Gaubatz, JDJ et al., Lipid Res.28 (1987) 69; Utermann, W. et al., FEBS Lett.154 (2) (1983) 357; Seman, WC. Cell Biol. 64 (1986) 999; Fless et al., J. Biol. Chem. 261 (19) (1986) 8712; Kraft, CJ et al., Clin. Invest. 80 (1987) 458). Seman et al. (Supra) found a carbohydrate content of 22.5% for Apo (a), and Fless et al. Also found a content of 54.1%. Apo (a) is closely related to plasminogen. Homology has been demonstrated by protein sequencing and cDNA cloning (McLean et al., Nature 300 (1987) 132). The proenzyme (zymogen) plasminogen contains five cysteine-rich sequences, each of which is 80-114 amino acids, the so-called "kringle", which are numbered from I to V. These relate to protein structures that are stabilized by three internal disulfide bridges between the first and sixth, the second and fourth, and the third and fifth cysteines. In addition, a serine protease region is located at the C-terminal end of Kringle V (Utermann et al., Supra). The signal sequence, kringle IV, kringle V and protease region contained in plasminogen are also present in Apo (a) and have great sequence homology with plasminogen. When kringle V of plasminogen is present in Apo (a), kringle IV is present in ten different variations (kringles 1-10 in Apo (a)). As far as the Apo (a) kringle 2 is concerned, it is also present in a different number (n = 1 to about 28) of identical copies. The resulting molecular weight differences classify Lp (a) and / or Apo (a) molecules into isoforms (Utermann et al .; Eaton et al., Proc. Natl. Acad. Sci. USA 84 (1987) 3224). The nomenclature currently distinguishes 6 and 37 isoforms (Lackner et al., J. Clin. Invest. 87 (1991) 2153-2161). The penultimate copy of Kringle IV probably contains another cysteine residue cross-linking Apo B-100 (Eaton et al., Supra). Based on the large homology between Apo (a) kringle and plasminogen kringle IV, Apo (a) is also presumed to have a lysine binding site. This could be confirmed by column chromatography on ricin sepharose. The kringle domain of plasminogen contains specific binding sites for fibrin, 2-antiplasmin and lysine. Kringles I-IV bind to lysine, whereby kringles I and V exhibit the greatest affinity (Eaton et al., Supra). Kringle structural elements are included in other classes of proteins, such as t-PA, urokinase, thrombin, factor VIII, and the like. Apo (a) contains the complete protease domain. It is enzymatically inactive and / or is not cleaved by streptokinase, urokinase or t-PA as in similar proteins. In the case of plasminogen, this is obtained by cleavage at Arginine 560 (Arg 560). However, the arginine residue essential for cleavage has been replaced by a serine in Apo (a) (Eaton et al., Supra). The carbohydrate component of Apo (a) is responsible for the hydrophilicity of this protein. It is approximately 45%, substantially higher compared to plasminogen (5%), and forms an important part of the antigenic properties of this protein. Mainly hexoses (galactose and mannose) and N-acetyl-D-galactosamine (GalNAc) and N-acetyl-D-glucosamine can be detected. The amount of hexose is similarly large in Apo (a), but the mannose content in LDL is greater than that of galactose. GalNAc is shown about 6 times better in LDL than in Apo (a). The neuraminic acid component can be cleaved by neuraminidase treatment, which apparently reduces the molecular weight of the molecule (Kraft et al., Supra; Utermann et al., J. Clin. Invest. 80 (1987) 458). The three-dimensional structure of Apo (a) has not yet been elucidated. To date, biophysical studies on recombinant Apo (a) (r-Apo (a)) are available. As shown by Phillips et al., Bioche mistry 32 (1993) 3722 (by Koschiensky et al., Biochemistry 30 (1991) 5044) for recombinant Apo (a), it has a Stokes radius of 94 ° and is highly asymmetric or Related to proteins that appear to be random coils. In electron microscopy, this has been observed as long, very flexible chains forming large open helical domains. And about 10% of the protein is present as polymer aggregates. The molecular weight is about 500 kD (Koschiensky et al., Supra). The molecular weight of human Apo (a) is between 280-800 kD depending on the isoform. All monomer molecules have a length of about 800 mm and consist of chains of 19 pliable domains, each with an average size of 42.1 mm. By comparison with the t-PA kringle structure revealed by X-ray crystallography, the Apo (a) region has a 28 x It could be presumed to be related to oblate ellipsoids with approximate dimensions of 30 x 11Å (see Phillips et al., Biochemistry 22 (1993) 3722, which illustrates the structure of Apo (a)). As already shown in other studies (Chiesa et al., J. Biol. Chem. 34 (1992) 24369; Lawn et al., Nature 360 (1992) 670; Breslow et al., Proc. Natl. Acad. Sci. USA 90 ( (1993) 8314), Apo (a), and the non-covalent association of Apo B-100 and / or all LDL molecules has also been successfully demonstrated here. Thus, r-Apo (a) is fixed to the LDL with at least one, and possibly several kringles, and spreads in a compact form in the surrounding space. This bond can be broken with 50 mM 6-aminohexanoic acid and is therefore non-covalent. In contrast, it appears to have predominantly ionic properties, as increasing the salt concentration reduces affinity. However, covalent bonds that actually exist as disulfide bridges in vivo do not appear to appear in vitro (Phillips et al., Supra). Like kringle 10-1, repeated kringle 2 has high homology to kringle IV of plasminogen. Unlike others, kringle 11 resembles kringle V of plasminogen (Eaton et al., Supra). From this homology it can be assumed that free Apo (a) and / or Lp (a) can intervene in the fibrinolytic system (Harpel et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3847). -3851; Logcalzo et al., Atherosclerosis 10 (1990) 240-245). Plasma concentrations, or Lp (a), are genetically determined and can be significantly eliminated from drug and diet susceptibility (Berg et al., Series Haematologica I (1968) 111), but concentrations above 300 μg / ml are considered significant. Measurement of plasma concentrations is of great diagnostic value because it represents an increased risk for congenital heart disease (CHD) (Berg et al., Clin. Genetics 8 (6) (1974) 230; Berg et al., Clin. Genetics 16). (1979) 374; Kostner et al., Atherosclerosis 38 (1 981) 151), thus requiring a more precise assessment of classic risk factors for CHD. The appearance of free Apo (a) in body fluids is a surprising finding. Apo (a) is linked to Apo B-100 with one or more disulfide bridges. However, these disulfide bridges are only closed in hepatocytes and are apparently not destroyed extracellularly (Alberts et al., The Cell (1983) 113-114). According to known methods, Lp (a) measurement is actually performed only by immunological methods. In this case, there are also RIA, radial immunodiffusion and nephelometry, but enzyme immunoassay (EIA) is the most important for quantitative measurement. Furthermore, qualitative detection of Lp (a) can be performed by immunoblot (Western blot). In this case, the antigens are first separated by gel electrophoresis (e.g., 4-15% polyacrylamide on a support thin film (Molinari et al., Lp (a) Phenotyping of the Phast System-Pharmacia; 55th Annual Meeting of the European Atherosclerosis Society (EAS), Brugge, Belgium, May 17-19, 1990); or 4% polyacrylamide / PEG (modified by Rig hetti et al., Electrophoresis 13 (1992) 587)). The proteins found on the gel are then transferred to a suitable membrane (eg, nitrocellulose) (Towbin et al., Proc. Natl. Acad. Sci. USA 76 (1979) 4350). The sample immobilized thereon can then be specifically detected by a suitable antibody-enzyme conjugate. Primarily, two antigens present on Lp (a), Apo (a) and Apo B-100, are suitable for detection. The solid-phase test system obtained from the combination of the anti-Apo (a) antibody in the conjugate and on the solid phase allows the measurement of Lp (a) and the simultaneous free Apo (a), It is not a distinction between both forms of Apo (a), nor a measurement of free Apo (a) form. In contrast, when the Apo B antibody is used in the conjugate, only Lp (a) can be detected. The reverse configuration, anti-Apo (a) in a conjugate with anti-Apo B on a solid phase, is only conditionally suitable for detecting Lp (a). This is because the presence of excess Apo B in LDL causes competition for binding to the solid phase antibody. With the addition of anti-Apo (a) antibodies, single-phase systems such as nephelometry and turbidometry form measurable, light-scattering or light-blocking immune complexes, Again, free Apo (a) and Apo (a) bound to Lp (a) cannot be distinguished. The discrepancy between the combined Lp (a) level and the relative risk of arteriosclerosis is also explained by considering the above model. There it is presumed that free Apo (a) is involved in the pathogenesis of atherosclerosis. However, as already mentioned above, there is no known method for detecting free Apo (a). Accordingly, an object of the present invention is to provide a method for measuring free apoprotein (a) in a human body fluid. This object is achieved by the subject of the present invention. The subject of the present invention is a method for determining the risk of lipoprotein metabolism disorders and / or atherogenesis present in a patient, characterized in that the measurement of free Apo (a) is carried out in a human body fluid. This is a diagnostic test method. Preferred embodiments of the method are the subject of claims 2-6. In the method for measuring free Apo (a) in a human body fluid according to the present invention, the Apo (a) bound to lipid is converted to the physical, physicochemical and / or chemical properties of lipid-bound Apo (a). Separate by known methods based on the differences. Preferably, the following methods: gel chromatography (based on the higher molecular weight of Apo (a) bound to lipid), binding to an affinity support (free Apo (a) different from Apo (a) bound to lipid) )) And the ability to bind on an affinity support), and for example, by known affinity chromatography methods. In particular, an immobilized lysine, an immobilized protein having a lysine present at an immobilized terminal position, for example, chemically attached to a support-bound aminocarboxylic acid, especially ε-aminocarboxylic acid, amine and / or heparin. The fixed one is used as an affinity support in these methods. In order to separate Apo (a) bound to lipids, it is also suitable to combine one or more methods which are the same or different from each other, especially at least one of the above-mentioned separation methods. Another subject of the present invention is a method for measuring free Apo (a) in a human body fluid containing free Apo (a) and Apo (a) bound to lipid, comprising the steps of: The above method, wherein free Apo (a) is measured by an immunoreaction with an antibody against a) but not against bound Apo (a). Preferred embodiments of the method are the subject of claims 8 and 9. The immune reaction with the antibody can be performed by a known method for this type of immune reaction. Preferably, the immune response is performed by an immunoassay using appropriately labeled antibodies. Antibodies are preferably labeled with enzymes, antigens, radionucleotides, fluorescent or luminescent sources. Therefore, according to the labeling method used for the antibody, the immunoassay can be performed by, for example, a commonly used enzyme immunoassay (EIA), particularly ELISA or radioimmunoassay (RIA). It has also been shown that immunoblotting (Western blot) is an immunological method, especially for the means of an immune reaction with an antibody according to the invention. According to this method, proteins from an electrophoresis gel are transferred to a fixed matrix (eg, a nitrocellulose filter) after separation, for example, in polyacrylamide gel electrophoresis. This is mainly done by electrical movement. The protein is then detected by an immunological reaction (eg, with a radioactive or enzyme-labeled antibody). The method of the present invention for measuring free Apo (a) involves the measurement of recombinant, modified and / or synthetic free Apo (a), and the determination of these in the form of aggregates of Apo (a) present. Suitable for measuring things. The method of the present invention, which can measure free Apo (a) present in human body fluids, is useful for the useful determination of lipoprotein metabolism disorders and the assessment of the risk of atherogenesis present in patients. Are suitable. Free Apo (a) is presumed to be involved in the pathogenesis of atherosclerosis, explaining the discrepancy between bound Lp (a) levels and the higher risk of arteriosclerosis. Unlike known measurement methods, it would be possible to make a valuable assessment based on the measurement of free Apo (a) present in human body fluids. Another subject of the present invention is a method for determining a lipoprotein metabolic disorder and assessing the risk of atherogenesis present in a patient, the method for measuring free Apo (a). Is performed in a body fluid obtained from a patient. Preferably, a blood, serum or plasma sample is used as the body fluid. The following examples are provided to illustrate the present invention in more detail, but the scope of the present invention is not limited to these embodiments. EXAMPLES In the following, preferred embodiments of the method of the present invention, which are the basis of the examples, are described by separating Apo (a) bound to lipids and measuring free Apo (a), and Preferred combinations of steps are described. Apo (a) bound in Lp (a) is separated for detection of free Apo (a). This is preferably done by solid phase methods (binding to antibodies that recognize only free Apo (a)), or by separation based on different molecular weights, different charges and other physicochemical properties. Electrophoresis is particularly suitable for separations based on different charges, and separation can be by molecular weight, such as by SDS electrophoresis, or by electrophoretic mobility, such as agar gel electrophoresis. Next, in a second step, free Apo (a) is made detectable. This is done in particular by a suitable immunological method. Immobilization of all proteins is performed, for example, on a nitrocellulose membrane, for example in a modified Western blot (Towbin et al., Supra). Thereafter, an antibody recognizing Apo (a) bound to, for example, free Apo (a) and LP (a) can be used. When the antibody is directly labeled (for example, with an enzyme), a site on the membrane on which Apo (a) is recognized as an antigen can be visualized by a color reaction. By comparison with a reaction with an anti-Apo B antibody, which is also performed directly but can be labeled with another marker enzyme or on a parallel blot strip , Free Apo (a) can be measured and, if necessary, can be quantitatively recorded by a known method. el et al., Clin. Chem. Acta 19/7 (1973) 737-739; Hieland et al., Clin. Chem. Acta1 9/10 (1973) 1139-1141; Heck et al., Clin. Chem. Lipid electrophoresis in Acta 23/78 (1977) 1296-1300; see Wieland et al., Innere Medizin (1978) 190-300)) is particularly suitable. According to this method, Lp (a) moves with LDL. It was found that free Apo (a) had higher electrophoretic mobility and therefore appeared before Lp (a). Detection of both forms of Apo (a) can be performed with an enzyme-labeled anti-Apo (a) antibody after transfer (by electrophoresis) to a protein binding membrane, preferably a nitrocellulose membrane. This converts the water-insoluble substrate at the site on the membrane where the antigen was recognized. In control blots, Apo B is similarly imaged with anti-Apo B antibody. Comparison of both blots shows a region with both delineable proteins and another region where only the anti-Apo (a) antibody response appeared. Example 1 Performing lipid electrophoresis 100 μl of plasma is mixed with an equal amount of agar medium and 10 μl of the mixture is applied to the gel. Thereafter, the application site is sealed with one drop of agar. After placing the gel in an electrophoresis chamber filled with electrophoresis buffer (62.1% 5,5′-diethylbarbituric acid sodium salt; 29.2% sodium citrate, 8.7% citrate, pH 8.6), 15 mA const Separate on gel for 180 minutes. Two application sites must be filled for each sample. Transfer A nitrocellulose membrane (Schleicher and Schuell) wetted with blotting buffer (25 mM Tris / HCl, 0.2 mM glycine, 20% v / v methanol) is placed on the gel. Migration occurs within 1 hour at 70 ° C. in the dry state. The membrane separated from the developing gel was blocked in 3% TBS / BSA for 1 hour, and washed with 4 times the volume of 1% TBS / BSA, followed by polyclonal rabbit-anti-Apo (a) and / or anti-Apo B Incubate overnight with -100 antibody. After washing with 4 volumes of 1% TBS / BSA, incubate with peroxidase (POD) -labeled-mouse-anti-rabbit antibody. Blot development is performed with diaminobenzidine. Comparison of the blots of Apo (a) and Apo B-100 shows free Apo (a). Results For CHD (= KHK) patients (No. 8), Western blotting of lipid electrophoresis showed two distinct bands that could be visualized with anti-Apo (a), and for healthy plasma donors (Q 10027) Is shown only once. LDL and Lp (a) can be stained with anti-Apo B. Thus, lower staining with anti-Apo (A) and anti-Apo B corresponds to Lp (a), whereas higher staining corresponds to free Apo (a). The blot pattern of the plasma donor (Q 10027) shows no Apo (a) band in the L DL region (stainable with anti-ApoB) and thus free Apo (a) Indicates that the double banding property (without β-mercaptoethanol) is removed. This leaves the LDL unchanged (staining with anti-Apo B). This can also be evaluated as detection of free Apo (a) (FIG. 2). Example 2 Apo (a) bound in Lp (a) is separated so that free Apo (a) can be detected. This can be done by separation according to molecular weight. For this, electrophoresis methods such as SDS electrophoresis are particularly suitable. After separation, the lipoproteins migrate to the protein-bound membrane where they are immobilized and subsequently detected immunologically. SDS electrophoresis is suitable as a method for separating lipoproteins from plasma according to molecular weight. As a result, Lp (a) moves together with LDL. Free Apo (a) has a higher electrophoretic mobility and is therefore seen before Lp (a). Detection of both Apo (a) forms can be performed with an enzyme-labeled anti-Apo (a) antibody after (electrophoretic) transfer to a protein-bound (nitrocellulose) membrane. This converts the water-insoluble substrate at the site above the membrane where the antigen is recognized. In control blots, Apo B is similarly imaged with anti-Apo B antibody. Comparison of both blots shows a region with both delineable proteins and another region where only the anti-Apo (a) antibody reacted. Performing electrophoresis The samples are separated on a polyacrylamide gel (T 4.2%, C 0.8%) with agarose solution (60%, 1.22 g in 100 ml). 750 mg of agarose was dissolved in 25 ml separation buffer (0.4% SDS, 1.5 mol / l Tris / HCl, pH 8.8) and 62.5 ml distilled water in a water bath (boiling), and 12.5 ml monomer stock solution (acrylamide 30% , N, N-methylenebisacrylamide 0.8%) and then cooled to 60 ° C. After the addition of 150 μl TEMED and 2.5 ml of a 10% ammonium persulphate solution, a bubble-free solution is obtained between two pre-warmed borosilicate glass plates. The polymerization takes place within 5 to 60 minutes. For the production of stacking gels, 0.5 ml monomer stock solution, 1.25 ml stacking gel buffer (0.4% SDS, 0.5 mol / l Tris / HCl, pH 6.8), 3.25 ml distilled water, 12 μl TEMED and 150 μl Mix 10% ammonium persulfate solution and layer on separation gel. The maximum sample volume at a gel thickness of 2.25 mm reaches about 100 μl. The electrophoresis is performed at 25 mA const./gel in an electrophoresis buffer of 25 mmol / l Tris / HCl, 0.2 mol / l glycine, 0.1% SDS, pH 8.0 for about 5 hours. Instead of this SD S-PAGE, the lipidophor gel can be separated by a modified SDS-PAGE (modified by Righetti et al., Electrophoresis 13 (1992) 587). Migration Migration is in this case preferably performed on a tank blot (25 mM Tris / HCl, pH 8.3, 20% v / v methanol, 16 hours, 15 ° C.). A nitrocellulose membrane (Schleicher and Schuell) wetted with blotting buffer is placed on the gel. The movement occurs in the direction of the anode. The membrane separated from the developing gel was blocked in 3% TBS / BSA for 1 hour, washed with 4 times the volume of 1% TBS / ESA, and then washed with polyclonal rabbit-anti-Apo (a) and / or anti-Apo. Incubate with B-100 antibody overnight. After washing with 4 volumes of 1% TBS / BSA, incubate with peroxidase (POD) -labeled-mouse-anti-rabbit antibody. Blot development is performed with diaminobenzidine. Comparison of the blots of Apo (a) and Apo B-100 shows free Apo (a). Results The macromolecular antigenic bands obtained with anti-Apo (a) and anti-Apo B antibodies correspond to Lp (a). Both proteins are linked by a disulfide bridge, which can be cleaved with β-mercaptoethanol under reducing conditions. This method also reveals another antigenic band further below Lp (a) with the anti-Apo (a) antibody. This corresponds to free Apo (a) present in plasma. Example 3 Two-dimensional electrophoresis can be performed by combining the methods described in Examples 1 and 2 so that free Apo (a) can be detected. This allows the Lipidophor Do. Detection of free Apo (a) is performed in the same manner as in Examples 1 and 2. For running, rebuffer the gel by placing it in sample buffer for 2 x 5 minutes. After removal of excess liquid, the gel can be polymerized with a collection gel in an SDS gel made as described. As an alternative to the SDS-PAGE described above, lipidephor gels can be separated by SDS-PAGE modified according to Righetti (Righetti et al., Electrophoresis 13 (1992) 587). Here, 1% PEG 20000 is added to the stock solution instead of agarose. Do the same for collection gels. Electrophoresis and detection of migrating and free Apo (a) is performed as described above. The membrane separated from the developing gel was blocked in 3% TBS / BSA for 1 hour, washed with 4 times the volume of 1% TBS / BSA, and then polyclonal POD-labeled rabbit-anti-Apo (a) and / or AP-labeled Incubate overnight with anti-Apo B-100 antibody. After washing in 4 volumes of 1% TBS / BSA, Neufuchsin or nitrotetrazolium blue (0.05% v / v 2 mol / l MgCl _Two 0.001% v / v nitrotetrazolium blue, 200 mmol 0.002% v / v 5-bromo-4-chloro-3-indoxyl phosphate Na salt in 1% TBS / BSA or 0.4% v / v levamisole, BCIP, Merck AG , Darmstadt, Germany), 0.02% v / v NaNO _Two The AP-labeled antibody is stained with 0.05% v / v naphthol-As-bistriphosphate in DMF, 0.005% v / v Neufuchsin in 0.1 mol / l Tris / HCl, pH 8.8). Thereafter, the POD component can be detected as described above. With this method, both proteins can be detected in one step. Free Apo (a) is detected there by POD reaction only. Double staining at one spot indicates the presence of both proteins (Apo (a) and Apo B). Results After two-dimensional electrophoresis, three spots are stained with anti-Apo (a) antibody. The (one-dimensional) immunologically displayed protein that migrates to the anode corresponds to Lp (a) with the highest molecular weight, while the lower molecular weight as a distinct double band is the free Apo (a) Corresponds to both isotypes. The stainable Apo (a) spots on the cathode have no Lp (a) present above and these can be clearly identified as free Apo (a). A form that gives the same electrophoretic mobility as Apo (a) together with B (without a covalent bond to the former) More Lp (a) spots disappear and only Apo (a) and Apo B are visualized with typical spots of each molecular weight (FIG. 4). Example 4 Apo (a) bound in Lp (a) is separated so that free Apo (a) can be detected. This is done here by gel filtration with molecular weight separation. Free Apo (a) l / cm ^Two / H, elute after Apo (a) at (200 mmol / l Tris / HCl, pH 8.0). Detection can be performed with a suitable enzyme or radioimmunoassay, and anti-Apo (a) antibody Perform gel filtration Do. Apply about 2.5 ml of sample according to the column size (26/70). Separation is 7.5 ml / cm ^Two Per hour in a 200 mmol / l Tris / HCl buffer, pH 8.0. EIA detection idelberg, Germany). Apo (a) is detected with anti-Apo (a) -Fab-POD conjugate. The five calibrators used show concentrations of 0, 150, 300, 500 and 800 μg / ml Lp (a) and the internal quality controls are 320-480 and 160-250 μg / ml. Standards and controls must be present as lyophilizates and reconstituted in incubation / wash buffer. Form incubation / wash buffer. The buffer used for the substrate reaction is H _Two O _Two Is an acetate buffer (pH 5.0). Use tetramethylbenzidine in ethanol / DMSO as substrate. The reaction is stopped with 2 mol / l sulfuric acid. Thereafter, the absorbance is measured and evaluated by an in-line computer. Results Proteins with Apo (a) antigenicity in EIA were eluted in the exclusion volume as well as in the internal volume. Complete Lp (a) is only found in the excluded volume in the separation matrix used. Thus, it is clear that the Apo (a) antigen is present as free, ie unbound Apo (a) (FIG. 5). Example 5 Apo (a) bound in Lp (a) must be separated in order to be able to detect free Apo (a). This is performed here by separation with the ability to bind lysine. Free Apo (a) has the ability to bind to lysine, which results in Apo (a) Under PB, pH 7.5, eluting with a linear gradient of ε-aminocaprionic acid), free Apo (a) elutes later than the apoprotein found in Lp (a). Detection can be performed with appropriate enzymes or p (a), Immuno) can be used on the solid phase as well as in the conjugate. Perform affinity chromatography Dilute the sample containing Lp (a) with the application buffer (1: 5) and apply to the column After washing with, this is eluted with a linear gradient of ε-aminocaprionic acid. Regenerate with at least 3 column volumes of wash buffer (1 mol / l NaCl, 0.05 mol / l ε-aminocaprionic acid, 0.05 mol / l PB, pH 7.5). Flow rate is 30 ml / cm ^Two / Hour. The progress of the chromatography is followed by a flow photometer with an in-line recorder. Detection by EIA Detection by EIA is performed as described in Example 4. Results Under appropriate application conditions as described above, Apo (a) bound into Lp (a) ) Can then be eluted with ε-ACA (FIG. 6). Example 6 Specific detection of Apo (a) not previously separated from Lp (a) in an enzyme immunoassay is described. For this purpose, antibodies are made available which can distinguish both forms. Such an antibody can only recognize the region of Apo (a), which is immobilized after binding Apo (a) to Apo B, for example, or cannot be sterically accessible Things. In testing, it is sufficient to use an antibody having such properties on a solid phase. Antibody Isolation Antibodies that specifically recognize free Apo (a) can be produced, for example, by targeted immunization with protein fragments. Protein fragments can be obtained by enzymatic or chemical degradation of Apo (a), by genetic engineering expression, or in the form of synthetic peptides. Such antibodies can be obtained by careful purification of polyclonal anti-Apo (a) plasma by affinity chromatography. As a result, all antibodies that recognize Lp (a) are removed, and antibodies that recognize free Apo (a) are concentrated. Test construction (a) Quantitative measurement of free Apo (a) For quantitative measurement of free Apo (a), the microtiter plate specifically recognizes free Apo (a) but Lp ( a) is coated with an unrecognized antibody. The test can be performed simultaneously or as a two-sided binding assay in series. An anti-Apo (a) antibody was used as the conjugate, again excluding Apo (a) and capable of recognizing LP (a). (b) Measurement of Lp (a) / Apo (a) ratioFor quantitative measurement of the ratio of bound Apo (a) to free Apo (a), the microtiter plate was tested for anti-Lp (a) Coating with antibody. After incubating the sample, it was detected with the two different conjugates and their corresponding substrates, ie, with the anti-Apo B-AP-conjugate, to quantify intact Lp (a) and the anti-Apo (a) -Quantify free Apo (a) by detection with POD-conjugate. BRIEF DESCRIPTION OF THE FIGURES FIG. 1: Apo B and free Ap in CHD (= KDK) patient (No. 8) and donor (Q10027) Rendering of Apo (a) and Apo B from donor (Q 10027) plasma below. FIG. 3 shows a Western blot after two-dimensional electrophoresis. Non by Molinari No. 8) sample). FIG. 4 shows a Western blot after two-dimensional electrophoresis. Return by Molinari Sample). Figure 5 shows the elution curve of Lp (a) isolation and the change in Apo (a) content after ultracentrifugation. 3 shows an elution curve of affinity chromatography.

────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Friedrich, Joseph             Germany Dee-69231 Rho             Emberg, Im Valmet 13 (72) Inventor Spawn, Renate             Germany Dee-72070 Tu             -Bingen, Holstadtstrasse               42

Claims (1)

[Claims] 1. Of free Apo (a) in human body fluids containing free Apo (a) and lipid-bound Apo (a) A measuring method, in which bound Apo (a) is separated and free Apo (a) is measured by a known method. The above method. 2. Apo (a) bound to lipids is separated by a method based on molecular weight. The method of claim 1, wherein 3. Apo (a) bound to lipids should be separated by gel chromatography. A method according to claim 1 or 2, characterized in that: 4. Separation of Apo (a) bound to lipids is based on the different binding capacities of free Apo (a). 2. The method according to claim 1, wherein the step is performed by bonding to a affinity support. the method of. 5. A separation method characterized in that the separation is carried out by affinity chromatography. The method according to claim 4. 6. Apo (a) bound to lipid is immobilized lysine, lysine immobilized support-bound amino Binding to immobilized proteins with carboxylic acids, amines and / or heparin The method according to claim 4, wherein the method is formed by: 7. Free Apo (a) in human body fluids containing free Apo (a) and lipid-bound Apo (a) The free Apo (a) is bound to free Apo (a) It is measured in an immune reaction with an antibody that is not directed against Apo (a). Said method. 8. Features immunoreaction by immunoassay using appropriate labeled antibody The method according to claim 7, wherein 9. The antibody is labeled with an enzyme, antigen, radionucleotide, fluorescent or luminescent source The method according to claim 7 or 8, wherein Ten. 11. The method for measuring recombinant, modified and / or synthetic free Apo (a). The method according to any of the above. 11． The method according to any one of claims 1 to 11, wherein the aggregated Apo (a) is measured. The method described. 12． Risk of lipoprotein metabolism disorders and / or atherogenesis present in patients It is a method of judging steepness, and for a body fluid obtained from a patient, free Apo ( before the measurement of a) is performed by the method according to any one of claims 1 to 11. Notation. 13. 13. The method according to claim 12, wherein the body fluid is a blood, serum or plasma sample. The method described.